Carp hemoglobin. II. The alkaline Bohr effect

J. C.W. Chien, K. H. Mayo

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The Bohr effect of carp hemoglobin has been determined by differential titration, by direct acid-base titration, and by calculation from precise oxygen equilibrium data over a wide pH variation. The results for the hemolysate and the two major components are essentially identical. At pH 6.9 in the vicinity of maximum cooperativity and maximum Bohr effect, the protein releases 3.7 protons in the absence of added ions. This Bohr curve above pH 7 is not changed by the presence of 0.05 M 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotri-ethanol (bis-Tris) buffer, but is changed below the pK of the bis-Tris amine, giving a maximum of 4.3 protons at pH 6.65. In 0.1 M phosphate, the maximum is increased to 6.1 protons and is shifted to pH 7.25. Addition of 1.4 mM P6-inositol lowers the magnitude of the Bohr effect and shifts its maximum to an even higher pH. At the limit of high pH (9.02), without buffer or in bis-Tris, there is a net uptake of about 0.5 proton upon oxygenation. The average heat of ionization of the Bohr groups is 5500 ± 800 cal. Even though chloride ion has a pronounced effect on the oxygenation properties of carp hemoglobin, it has a small influence on the Bohr effect up to 0.5 M NaCl, the magnitude of the Bohr effect is reduced by approximately 30%. Acid base titrations give three to four oxygen-linked groups for carp hemoglobin in water; this is increased to about six groups in 2.5 M NaCl. The results suggest that carp hemoglobin is functionally versatile and may provide one way to regulate its CO2 transport via heterotropic allosteric interactions. In phosphate buffer at the pH valve where carp hemoglobin is strongly cooperative, the proton release is linear with respect to ligand saturation. Lowering the cooperativity by either an increase or decrease in pH results in nonlinear relationships.

Original languageEnglish (US)
Pages (from-to)9800-9806
Number of pages7
JournalJournal of Biological Chemistry
Issue number20
StatePublished - 1980
Externally publishedYes


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